Cholinergic Receptor Function Research Articles

M4 muscarinic receptors regulate the dynamics of cholinergic and dopaminergic neurotransmission: relevance to the pathophysiology and treatment of related CNS pathologies.

Dopaminergic dysfunction is an important pathogenetic factor for brain pathologies such as Parkinson's disease, ADHD, schizophrenia, and addiction as well as for metabolic disorders and anorexia. Dopaminergic neurons projecting from the midbrain to forebrain regions, such as the nucleus accumbens and the prefrontal cortex, regulate motor and cognitive functions and coordinate the patterned response of the organism to sensory, affective, and rewarding stimuli. In this study, we showed that dopaminergic neurotransmission is highly dependent on M4 cholinergic muscarinic receptor function. Using in vivo microdialysis, we found elevated dopamine (DA) basal values and enhanced DA response to psychostimulants in the nucleus accumbens of M4 knockout mice. We also demonstrated impaired homeostatic control of cholinergic activity that leads to increased basal acetylcholine efflux in the midbrain of these animals. Thus, loss of M4 muscarinic receptor control of cholinergic function effectuates a state of dopaminergic hyperexcitability. This may be responsible for pathological conditions, in which appetitive motivation as well as affective and cognitive processing is impaired. We propose that M4 receptor agonists could represent an innovative strategy for the treatment of pathologies associated with hyperdopaminergia.

Ontogeny of cholinergic regulation of fetal upper gastrointestinal motility

Objective: In the fetal rabbit immediately prior to birth (day 30; 0.97 gestation), intragastric atropine suppresses upper gastrointestinal (GI) motility, indicating that cholinergic receptors are expressed and functional at birth. To explore the developmental timing of upper GI cholinergic receptor function, we assessed the effect of intragastric atropine administration in rabbit fetuses during the last 10% of gestation. Methods: Pregnant rabbits were studied at day 27, day 28 and day 29 of their normal 31-day gestation. In each litter, two fetuses were selected as study fetuses and two as control fetuses. Under ultrasound guidance, fluorescein and either atropine (0.04 μg/g fetal body weight) or normal saline were injected into the fetal stomach. Two hours after injection, fetuses were delivered and the small intestine was harvested. The per cent motility was calculated as the fluorescein travel distance, which was measured by ultraviolet light optical density, divided by the total small intestinal length. Results: Fetal body weight, small intestinal length and per cent motility increased from day 27 to day 29 (p < 0.01). There were no differences in fetal body weight and small intestinal length between atropine and control groups. Atropine significantly decreased per cent motility (versus control values) in fetuses at day 29 and day 28 (56.1 ± 13.5 vs. 66.1 ± 11.7% and 59.7 ± 15.6 vs. 68.3 ± 11.7%, respectively; p < 0.05), but not at day 27 (52.4 ± 12.9 vs. 52.8 ± 11.2%). Conclusions: These results indicate that upper GI functional cholinergic receptors develop between 0.87 and 0.90 of rabbit gestation. Extrapolation to human development suggests that reduced GI motility in preterm human infants results, in part, from immature GI cholinergic receptors.

Octopaminergic modulation of synaptic transmission between an identified sensory afferent and flight motoneuron in the locust

The role of the biogenic amine octopamine in modulating cholinergic synaptic transmission between the locust forewing stretch receptor neuron (fSR) and the first basalar motoneuron (BA1) was investigated. The amines 5-hydroxytryptamine (5-HT, serotonin) and dopamine were also studied. Bath application of octopamine, 5-HT, and dopamine at concentrations of 10(-4) M reversibly decreased the amplitude of monosynaptic excitatory postsynaptic potentials (EPSPs) evoked in BA1 by electrically stimulating the fSR axon. These effects occurred without any detectable change in either input resistance or membrane potential of BA1. The amines also reversibly decreased the amplitude of responses to acetylcholine (ACh) pressure-applied to the soma of BA1. The muscarinic antagonist scopolamine (10(-6) M) had no significant effect on the octopamine-induced decrease in ACh responses. These observations suggest that these amines potentially could physiologically depress cholinergic transmission between fSR and BA1, at least in part, by altering nicotinic rather than muscarinic cholinergic receptor function. Although the octopaminergic agonists naphazoline and tolazoline both mimicked the actions of octopamine, the receptor responsible for octopamine-mediated modulation could not be characterized since amine receptor antagonists tested on the preparation had complex actions. Confocal immunocytochemistry revealed intense octopamine immunoreactivity in the anterior lateral association center, thus confirming the presence of octopamine in neuropil regions containing fSR/BA1 synapses and therefore supporting a role for this amine in the modulation of synaptic transmission between the fSR and BA1. 5-HT-immunoreactivity, conversely, was concentrated within the ventral association centers; very little staining was observed in the dorsal neuropil regions in which fSR/BA1 synapses are located.

Ontogeny of cholinergic regulation of fetal upper gastrointestinal motility

Objective: In the fetal rabbit immediately prior to birth (day 30; 0.97 gestation), intragastric atropine suppresses upper gastrointestinal (GI) motility, indicating that cholinergic receptors areexpressed and functional at birth. To explore the developmental timing of upper GI cholinergic receptor function, we assessed the effect of intragastric atropine administration in rabbit fetuses duringthe last 10% of gestation.Methods: Pregnant rabbits were studied at day 27, day 28 and day 29 of their normal 31-day gestation. In each litter, two fetuses were selected as study fetuses andtwo as control fetuses. Under ultrasound guidance, fluorescein and either atropine (0.04 μg/g fetal body weight) or normal saline were injected into the fetal stomach. Two hours after injection, fetuseswere delivered and the small intestine was harvested. The per cent motility was calculated as the fluorescein travel distance, which was measured by ultraviolet light optical density, divided by the totalsmall intestinal length.Results: Fetal body weight, small intestinal length and per cent motility increased from day 27 to day 29 (p < 0.01). There were no differences in fetal bodyweight and small intestinal length between atropine and control groups. Atropine significantly decreased per cent motility (versus control values) in fetuses at day 29 and day 28 (56.1 ± 13.5 vs.66.1 ± 11.7% and 59.7 ± 15.6 vs. 68.3 ± 11.7%, respectively; p < 0.05), but not at day 27 (52.4 ± 12.9 vs. 52.8 ± 11.2%).Conclusions: These resultsindicate that upper GI functional cholinergic receptors develop between 0.87 and 0.90 of rabbit gestation. Extrapolation to human development suggests that reduced GI motility in preterm human infants results,in part, from immature GI cholinergic receptors.

Beta-Adrenergic modulation of muscarinic cholinergic receptor expression and function in developing heart.

Imbalances of beta-adrenoceptor (beta-AR) and muscarinic ACh receptor (mAChR) input are thought to underlie perinatal cardiovascular abnormalities in conditions such as sudden infant death syndrome. Administration of isoproterenol, a beta(1)/beta(2)-AR agonist, to neonatal rats on postnatal days (PN) 2-5 caused downregulation of cardiac m(2)AChRs and a corresponding decrement in their control of adenylyl cyclase activity. Terbutaline, a beta(2)-selective agonist that crosses the placenta and the blood-brain barrier, was also effective when given either on PN 2-5 or during gestational days 17-20. Terbutaline failed to downregulate brain m(2)AChRs, even though it downregulated beta-ARs; beta-ARs and m(2)AChRs are located on different cell populations in the brain, but they are on the same cells in the heart. Destruction of catecholaminergic neurons with neonatal 6-hydroxydopamine upregulated cardiac but not brain m(2)AChRs. These results suggest that perinatal beta-AR stimulation shifts cardiac receptor production away from the generation of m(2)AChRs so that the development of sympathetic innervation acts as a negative modulator of cholinergic function. Accordingly, tocolytic therapy with beta-AR agonists may compromise the perinatal balance of adrenergic and cholinergic inputs.

α10: A determinant of nicotinic cholinergic receptor function in mammalian vestibular and cochlear mechanosensory hair cells

We report the cloning and characterization of rat alpha10, a previously unidentified member of the nicotinic acetylcholine receptor (nAChR) subunit gene family. The protein encoded by the alpha10 nAChR subunit gene is most similar to the rat alpha9 nAChR, and both alpha9 and alpha10 subunit genes are transcribed in adult rat mechanosensory hair cells. Injection of Xenopus laevis oocytes with alpha10 cRNA alone or in pairwise combinations with either alpha2-alpha6 or beta2-beta4 subunit cRNAs yielded no detectable ACh-gated currents. However, coinjection of alpha9 and alpha10 cRNAs resulted in the appearance of an unusual nAChR subtype. Compared with homomeric alpha9 channels, the alpha9alpha10 nAChR subtype displays faster and more extensive agonist-mediated desensitization, a distinct current-voltage relationship, and a biphasic response to changes in extracellular Ca(2+) ions. The pharmacological profiles of homomeric alpha9 and heteromeric alpha9alpha10 nAChRs are essentially indistinguishable and closely resemble those reported for endogenous cholinergic eceptors found in vertebrate hair cells. Our data suggest that efferent modulation of hair cell function occurs, at least in part, through heteromeric nAChRs assembled from both alpha9 and alpha10 subunits.

Myocardial adrenergic and cholinergic receptor function in hypoxia: correlation with O(2) transport in exercise.

The time course of changes in rat myocardial alpha(1)- and beta-adrenoceptors and of muscarinic cholinergic (M-Ach) receptor characteristics was studied parallel with the changes in exercise systemic O(2) transport during a 21-day period of hypoxia (barometric pressure 380 Torr) to assess the effects of receptor modification during acclimatization on maximal exercise capacity. Hypoxia resulted in polycythemia, pulmonary hypertension, right ventricular hypertrophy, and transient left ventricular weight loss. Maximal O(2) consumption at 30 min of hypoxia was reduced to 60% of the normoxic value and remained unchanged. This was partly due to a gradual decrease in maximal cardiac output and heart rate (HR(max)), which offset the increase in blood O(2) content. HR(max) correlated positively (r = 0.994) with beta-adrenoceptor density and negatively (r = -0.964) with M-Ach-receptor density, suggesting that HR(max) reduction results from intrinsic changes in myocardial receptor characteristics leading to reduced responses to adrenergic stimulation and elevated responses to cholinergic stimulation. alpha-Adrenoceptor density in both ventricles increased initially to eventually fall below normoxic values. The dissociation between the different patterns of right and left ventricular weight and the similar pattern of alpha-adrenoceptor change in both ventricles do not support a role for these receptors on right ventricular myocardial hypertrophy.

Cholinergic and serotonergic alterations in the rat hippocampus following trimethyltin exposure and fetal neural transplantation

Trimethyltin (TMT) apart from causing cholinergic denervation of the hippocampus, damages the serotonergic inputs into the hippocampus as well. In the present study, fetal cholinergic and serotonergic rich neuronal populations from septal and raphe regions, respectively, were transplanted alone or in combination (as co-grafts) in the hippocampus of TMT exposed rats. Neurotransmitter receptor binding and neurotransmitter levels were assayed 6 months post-transplantation. Fetal septal transplants (rich in cholinergic neurons) significantly restored the deficits in cholinergic (muscarinic) receptor binding and acetylcholinesterase activity caused by TMT exposure. Raphe transplants (rich in serotonergic neurons) restored the deficit in serotonergic receptor binding and serotonin levels caused by TMT. Co-grafts of fetal raphe and septal neurons restored both the cholinergic (muscarinic) and serotonergic receptor functions. The results suggest that co-grafting technique could provide a better restoration of functional deficits when more than one type of neuronal population is damaged.

Alterations of striatal cholinergic receptors after lesioning of the substantia nigra

Dopamine deficiency syndrome is known to cause cholinergic hyperactivity. Therefore, it was hypothesized that the said phenom- enon may be due to enhanced cholinergic receptor functions. In the present study we examined the changes in striatal dopa- minergic and cholinergic receptors in unilateral substantia nigra lesioned rats that showed vigorous ipsilateral rotation (total turns >300) in response to apomorphine (1 mg kg -1 ip). [ 3H] Spiperone ( 3H -SP) and [ 3 H]-quinuclidinyl benzilate ( 3H -QNB) bindings were performed in the striata of the lesioned animals. There was no significant difference in the dissociation equilibrium constant values (K d) between the lesioned and non-lesioned sides. However, a significant difference in the maximum receptor density (B max ) of both [ 3H]-SP and [ 3H]-QNB bindings was observed between the lesioned and non-lesioned sides. The B max of [ 3H]-SP binding was significantly decreased on the lesioned side, whereas the B max of the [ 3H]-QNB binding was significantly increased. These results support the hypothesis that deficiencies of the dopaminergic system cause overactivity of the cholinergic system in the striatum.

Effects ofin Uteroand Lactational Exposure of the Laboratory Rat to 2,4,2′,4′- and 3,4,3′,4′-Tetrachlorobiphenyl on Dopamine Function

Offspring of Sprague–Dawley derived dams were exposed to either 2,4,2′,4′-tetrachlorobiphenyl (TCB) (1, 10, or 20 mg/(kg·day)) or 3,4,3′,4′-TCB (0.1 or 1 mg/(kg·day)) from gestational Day 6 through weaning by providing the dams with cookies adulterated with the appropriate amount and type of PCB. Male and female offspring were sacrificed on postnatal Days 35, 60, and 90, and brain concentrations of dopamine and its metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, were determined in the frontal cortex, caudate nucleus and substantia nigra by high-performance liquid chromatography with electrochemical detection.In uteroand lactational exposure to 3,4,3′,4′-TCB resulted in significant elevations in concentrations of dopamine in the frontal cortex, and of dopamine and its metabolites in the substantia nigra that persisted into adulthood. In contrast,in uteroand lactational exposure to 2,4,2′,4′-TCB resulted in significant decreases in concentrations of dopamine in the frontal cortex and caudate nucleus that also persisted into adulthood. We suggest that the reductions in brain dopamine concentrations are a consequence ofortho-substituted PCB congener-induced inhibition of the synthesis of dopamine during critical periods of development acting, perhaps, in concert with PCB-induced changes in cholinergic receptor function. On the other hand, the persistent elevations in brain dopamine and metabolite concentrations following perinatal exposure to 3,4,3′,4′-TCB may be mediated by alterations in steroid hormone function during key developmental periods.

Biological functions of keratinocyte cholinergic receptors.

Human epidermal keratinocytes possess cholinergic enzymes, which synthesize and degrade acetylcholine, and express both nicotinic and muscarinic classes of cholinergic receptors on their cell surfaces. These receptors bind acetylcholine and initiate cellular response. The presence in keratinocytes of a functional cholinergic system suggests a role for acetylcholine in most, if not all, aspects of keratinocyte function. Autocrine and paracrine acetylcholine are required to sustain the viability of keratinocytes in vitro, and cholinergic drugs can alter keratinocyte proliferation, adhesion, migration, and differentiation. Acetylcholine employs calcium as a mediator for its effects on keratinocytes. In turn, changes in calcium concentration may affect expression and function of keratinocyte cholinergic enzymes and cholinergic receptors. At different stages of their differentiation, keratinocytes may demonstrate unique combinations of cholinergic enzymes and cholinergic receptor types. This would allow basal, prickle, and granular keratinocytes to respond to acetylcholine differently, in accordance with their functions at each stage of keratinocyte development in epidermis.

Modulation of hippocampal norepinephrine release by cholinergic agonists is altered by AF64A lesion.

The effect of lesioning hippocampal cholinergic neurons with the neurotoxin AF64A on the ability of cholinergic agonists to modulate stimulation-induced release of 3H-norepinephrine (NE) from rat hippocampal slices was studied. Rats received intracerebroventricular injections of either AF64A (ethylcholine mustard aziridinium, 2 nmol) or vehicle (sham operated). Six weeks after treatment, release of 3H-NE evoked by electrical stimulation (2 Hz, 2 min) in the presence or absence of cholinergic agonists and/or antagonists was measured. Activation of M2 receptors with oxotremorine (in the presence of the M1 antagonist pirenzepine) caused a small inhibition of NE release, which was abolished in hippocampi from AF64A-treated rats. The Kd for high-affinity binding of the selective M2 ligand [3H] AF-DX 384 was increased 10-fold in lesioned tissues. The M1 selective agonist McN-A-343 produced a significant enhancement of NE release, which was unchanged by AF64A lesion. Binding studies with [3H] pirenzepine showed no change in the affinity or number of M1 receptors. Nicotine also caused a significant enhancement of evoked NE release, but this effect was markedly reduced in tissues from AF64A-treated rats. AF64A treatment caused a twofold decrease in the number of [3H] nicotine binding sites. This study suggests that long-term lesion of hippocampal cholinergic neurons with AF64A alters the function of postsynaptic muscarinic M2 and nicotinic cholinergic receptors that modulate the release of NE in the hippocampus.

Dysautonomia in dogs: a retrospective study.

Dysautonomia was diagnosed in 11 young (median age, 14-months), predominantly medium- to large-breed dogs from 1988 to 1995. Clinical signs caused by autonomic dysfunction of the urinary, alimentary, and ocular systems were most common. Dysuria, mydriasis, absence of pupillary light reflexes, decreased tear production, dry mucous membranes, weight loss, and decreased anal tone were present in over 75% of affected dogs. Ocular pharmacological testing with a dilute (0.1%) solution of pilocarpine was used to demonstrate iris sphincter receptor function in all dogs. A low-dose (0.0375 mg/kg s.c.) bethanechol test and pharmacological testing with phenylephrine and epinephrine also demonstrated cholinergic and adrenergic receptor function in 4 dogs. All dogs died or were euthanized as a results of autonomic dysfunction. Neuronal depletion, with associated gliosis and minimal inflammation were noted histologically in the autonomic ganglia of each dog. The pelvic, ciliary, celiac, cranial cervical, and cranial and caudal mesenteric ganglia were affected in all dogs. The cause of autonomic failure in these dogs was not determined.

Oligotide, a new single-stranded oligodeoxyribonucleotide, preserves postsynaptic β-adrenergic and cholinergic receptor functions in rabbit hearts after acute infarction

Acute myocardial infarction was induced in New Zealand male albino rabbits. 3 days after surgery, the mortality rate and plasma CPK activity were reduced by 64% and 66% respectively by intravenous infusion of Oligotide (32 mg/kg/h for 6h), a single-stranded oligodeoxyribonucleotide of mammalian origin. Perfused hearts, obtained from Oligotide-treated rabbits 3 days after surgery, showed a strength of contraction in the range of that of hearts of sham-operated animals and an ameliorated postsynaptic β-adrenergic and cholinergic receptor function. In addition, in these hearts, “ex vivo” treatment with Oligotide resulted in almost complete preservation of both adrenergic and cholinergic responses to their specific agonists. These data suggest that Oligotide by protecting the hearts from the ischemic damage and possibly by restricting left ventricular tissue necrosis may have normalized the biological responses of this organ to isoproterenol and preserved the integrity of coronary endothelial-dependent relaxant function.

Cholinergic regulation of [Ca2+]i during cell division and differentiation in the mammalian retina

Transmitter-like molecules are thought to influence many aspects of neuronal development, often by regulating the levels of intracellular calcium. Using the Ca2+ sensitive dye, fura-2, this study demonstrates that in the rabbit retina, ACh analogs stimulate a rise in cytosolic free Ca2+ concentration ([Ca2+]i) in many cell types, and in cells at various stages of differentiation during embryonic and neonatal development. The elevation in [Ca2+]i in cells within the ventricular zone (VZ) resulted primarily from the activation of muscarinic receptors. By contrast, the cholinergic regulation of [Ca2+]i of ganglion cells and amacrine cells, cell types which migrate to their final destinations early in fetal life, was largely mediated by nicotinic receptors. The muscarinic response of the VZ cells was mediated by the M1, rather than the M2-type of muscarinic receptor. This response was abolished in the absence of extracellular Ca2+ and in the presence of NiCl2, but it was not affected by verapamil or omega-conotoxin, thus suggesting that while Ca2+ influx occurred, it did not involve L- and N-type voltage-gated Ca2+ channels. The muscarinic response in the VZ disappeared at the end of the period of cell division in the retina, just prior to eye opening. By contrast, nicotinic-induced changes in [Ca2+]i in ganglion cells and amacrine cells persisted throughout development. Since previous studies have implicated that the precursors of ganglion cells and amacrine cells also possess muscarinic receptors (Yamashita and Fukuda, 1993), the concomitant emergence of different functional cholinergic receptor (AChR) subtypes with differentiation in vivo suggests that ACh may play diverse and temporally regulated roles in the developing retina.

Lesion with the neurotoxin AF64A alters hippocampal cholinergic receptor function

The effect of selective lesion of cholinergic inputs to the hippocampus on the function of hippocampal cholinergic receptors was examined. Hippocampal cholinergic neurons were lesioned in the rat by administration of the selective cholinergic neurotoxin AF64A (ethylcholine mustard azirtdinium). Cholinergic receptor function was examined by assessing the ability of cholinergic agonists and antagonists to modulate the evoked release of radiolabelled acetylcholine (ACh) from hippocampal slices. Nicotine enhanced release, with a bell-shaped dose-response curve. The dose-response curve and EC 50 for nicotine was shifted 10-fold to the left in lesioned rats, suggesting an increased sensitivity to nicotine. However, there were no differences in either the number or affinity of nicotinic receptors as determined with binding studies. The muscarinic agonist oxotremorine inhibited the evoked release of ACh in control tissues, but had much less effect in AF64A-lesioned tissues. Binding to the M1 receptor subtype was not changed. However, the K d for binding to the high affinity subtype of the M2 receptor was increased 10-fold, suggesting that the receptor has become less sensitive to stimulation. Loss of M2 function may allow an increase in the effect of stimulating nicotinic receptors that modulate ACh release.

Tryptamine induces phosphoinositide turnover and modulates adrenergic and muscarinic cholinergic receptor function in cultured cerebellar granule cells.

Tryptamine dose-dependently increased phosphoinositide (PI) hydrolysis by approximately fourfold in primary cultures of rat cerebellar granule cells (EC50 = 56 microM). The PI response stimulated by tryptamine was dependent on the presence of extracellular Ca2+ and Na+. Tryptamine-induced PI breakdown could be partially inhibited by pretreatment with 4 beta-phorbol 12-myristate 13-acetate but not pertussis toxin. The presence of tryptamine markedly attenuated PI responses induced by norepinephrine (NE) and carbachol, with no apparent effect on the responses to 5-hydroxytryptamine and glutamate. The inhibition of NE- and carbachol-induced PI turnover by tryptamine was dose dependent with IC50 values of approximately 0.4 and approximately 2.5 mM, respectively. Pretreatment of cells with tryptamine (0.5 mM) also attenuated NE- and carbachol-induced PI turnover, but failed to affect 5-hydroxytryptamine- and glutamate-induced responses. Furthermore, ketanserin, atropine, and prazosin did not have any effect on inositol phosphate formation induced by tryptamine. These observations indicate that tryptamine markedly increased Ca(2+)- and Na(+)-dependent PI turnover in cerebellar neurons and selectively inhibited NE- and carbachol-induced PI hydrolysis.

Two distinct receptors are activated by arecoline on cockroach sixth abdominal ganglion DUM neurones

ABSTRACT During the last decade, extensive biochemical and electrophysiological studies have been performed to characterize the pharmacological properties of cholinergic receptors of insect neurone somata (Sattelle, 1985; Breer and Sattelle, 1987; Benson, 1992). Although most of these studies have been focused on the pharmacological characterization of the cell body nicotinic receptors (Lane et al. 1982; Harrow and Sattelle, 1983; David and Sattelle, 1984; Benson, 1992), evidence for a population of acetylcholine (ACh) receptors exhibiting muscarinic and ‘mixed’ (nicotinic/muscarinic) properties has been reported on insect neurone somata (Benson and Neumann, 1987; Knipper and Breer, 1988; Benson, 1989, 1992, 1993; Trimmer and Weeks, 1989; David and Pitman, 1990, 1992, 1993). One group of insect neurones, called dorsal unpaired median (DUM) neurones, expresses functional cholinergic receptors that differ from the classic vertebrate ACh receptors in that, although DUM neurones were sensitive to ACh, the nicotinic and/or muscarinic nature of these receptors has not been resolved since the cholinergic antagonists α-bungarotoxin and atropine were relatively ineffective in blocking the ACh response (Goodman and Spitzer, 1980; Lane et al. 1982; Lapied et al. 1990). However, using a range of cholinergic antagonists known to be selective for vertebrate ACh receptors, it has recently been possible to demonstrate the diversity of functional ACh receptors; these include nicotinic, ‘mixed’ and muscarinic receptors mediating specific responses elicited by different cholinergic agonists, such as nicotine, muscarine, oxotremorine and McN-A-343, on both isolated and in situ DUM neurones (Lapied et al. 1990, 1992; Bai et al. 1992). Extending these studies, we report in this paper that another cholinergic ligand, arecoline, which is known to act preferentially as a muscarinic agonist in vertebrate preparations (Wess et al. 1990), induced a complex response mediated by both nicotinic and muscarinic receptors on DUM neurones.

Regulation of hippocampal muscarinic receptor function by chronic nerve growth factor treatment in adult rats with fimbrial transections

Effects of chronic intraventricular administration of recombinant human nerve growth factor on hippocampal muscarinic receptor densities and muscarinic receptor-linked second messenger systems were determined in adult rats 21 days following partial or full unilateral fimbrial transections. First, autoradiographic analysis of muscarinic receptors was carried out using [ 3H]quinuclidinyl benzilate for total muscarinic receptors, [ 3H]pirenzepine for M1 receptors and [ 3H]AF-DX 384 for M2 receptors. Partial fimbrial transections did not significantly alter the density of these muscarinic receptor populations in the dorsal or ventral hippocampus and there was no effect of chronic (1 /gmg every other day, 21 days) recombinant human nerve growth factor treatment. In contrast, in animals receiving full fimbrial transections which by themselves did not alter muscarinic receptor density, recombinant human nerve growth factor treatment increased the density of [ 3H]quinuclidinyl benzilate binding sites. M1 receptors, and M2 receptors by approximately 40% in the CA1 region. Secondly, we determined the effect of chronic recombinant human nerve growth factor treatment on muscarinic receptor-mediated second messenger production in rats with either partial or full unilateral fimbrial transections. In partially fimbriectomized rats, oxotremorine-induced inositol triphosphate production by hippocampal slices was increased by 81% on the lesioned side of animals treated with a control protein. This lesion-induced supersensitivity of M1 muscarinic receptor function was prevented by chronic recombinant human nerve growth factor treatment. In recombinant human nerve growth factor-treated animals, inositol triphosphate production was similar to values on unlesioned control sides. The muscarinic receptor-mediated increase in cyclic GMP levels was not altered by fimbrial transections or recombinant human nerve growth factor treatment. In animals with full unilateral fimbrial transections, oxotremorine-induced inositol triphosphate production was increased by 99% on the lesioned side of animals treated with a control protein and treatment with recombinant human nerve growth factor did not alter this denervation-induced supersensitivity of muscarinic receptor transduction signal. Chronic recombinant human nerve growth factor treatment did not affect the levels of inositol triphosphate on the contralateral unlesioned side of either partial or full fimbriectomized animals. Earlier studies indicate that chronic nerve growth factor treatment increases the presynaptic function of hippocampal cholinergic neurons surviving partial fimbrial transections. The findings of the present study indicate that these presynaptic effects translate into functional changes at the level of postsynaptic muscarinic receptors in the hippocampus. In addition, our studies suggest that a presynaptic cholinergic component is necessary to observe recombinant human nerve growth factor-induced enhancements of cholinergic receptor function in the hippocampus, since in animals receiving full unilateral transections muscarinic receptor-mediated responses are not attenuated following recombinant human nerve growth factor treatment.

Effects of chronic nicotine treatment on the accumulation of [3H]tetraphenylphosphonium by cerebral cortical synaptosomes.

Chronic exposure of rats to nicotine increases the number of [3H]nicotine binding sites in the brain; however, it is not clear whether nicotinic cholinergic receptor function is altered as well. In this study, we have used [3H]tetraphenylphosphonium as a probe of synaptosomal membrane potential to investigate whether exposure to nicotine in vivo alters the ability of cerebral cortical synaptosomes to maintain a potential difference and to depolarize in response to in vitro nicotine. Treatment of rats for 14 days with 0.475 mg of nicotine base/day via subcutaneously implanted minipumps resulted in a decrease in the synaptosomal accumulation of [3H]tetraphenylphosphonium in physiological buffer, corresponding to a decrease in estimated membrane potential from -55 mV to -50 mV. The onset of the decrease in membrane potential occurred after 7 days of in vivo nicotine treatment and was significantly correlated with an increase in [3H]nicotine binding to cerebral cortical synaptosomal (P2) membranes. Nicotine, at in vitro concentrations of 3-1,000 microM, decreased [3H]tetraphenylphosphonium accumulation in cerebral cortical synaptosomes from control animals. When compared to accumulation in buffer alone, in vitro nicotine and other nicotinic agonists did not significantly decrease [3H]tetraphenylphosphonium accumulation in cerebral cortical synaptosomes prepared from rats treated with nicotine in vivo. These studies provide evidence that chronic treatment with nicotine results in an average lower membrane potential in cerebral cortical synaptosomes and in functional down-regulation of the depolarization response to nicotinic cholinergic receptor stimulation.